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Clinical Trials: Immunotherapy

Phase I Clinical Trial of Combination Propranolol and Pembrolizumab in Locally Advanced and Metastatic Melanoma: Safety, Tolerability, and Preliminary Evidence of Antitumor Activity

Shipra Gandhi, Manu R. Pandey, Kristopher Attwood, Wenyan Ji, Agnieszka K. Witkiewicz, Erik S. Knudsen, Cheryl Allen, Joseph D. Tario, Paul K. Wallace, Carlos D. Cedeno, Maria Levis, Suzanne Stack, Pauline Funchain, Joseph J. Drabick, Mark J. Bucsek, Igor Puzanov, Hemn Mohammadpour, Elizabeth A. Repasky and Marc S. Ernstoff
DOI: 10.1158/1078-0432.CCR-20-2381 Published January 2021

Abstract

Purpose: Increased β-adrenergic receptor (β-AR) signaling has been shown to promote the creation of an immunosuppressive tumor microenvironment (TME). Preclinical studies have shown that abrogation of this signaling pathway, particularly β2-AR, provides a more favorable TME that enhances the activity of anti–PD-1 checkpoint inhibitors. We hypothesize that blocking stress-related immunosuppressive pathways would improve tumor response to immune checkpoint inhibitors in patients. Here, we report the results of dose escalation of a nonselective β-blocker (propranolol) with pembrolizumab in patients with metastatic melanoma.

Patients and Methods: A 3 + 3 dose escalation study for propranolol twice a day with pembrolizumab (200 mg every 3 weeks) was completed. The primary objective was to determine the recommended phase II dose (RP2D). Additional objectives included safety, antitumor activity, and biomarker analyses. Responders were defined as patients with complete or partial response per immune-modified RECIST at 6 months.

Results: Nine patients with metastatic melanoma received increasing doses of propranolol in cohorts of 10, 20, and 30 mg twice a day. No dose-limiting toxicities were observed. Most common treatment-related adverse events (TRAEs) were rash, fatigue, and vitiligo, observed in 44% patients. One patient developed two grade ≥3 TRAEs. Objective response rate was 78%. While no significant changes in treatment-associated biomarkers were observed, an increase in IFNγ and a decrease in IL6 was noted in responders.

Conclusions: Combination of propranolol with pembrolizumab in treatment-naïve metastatic melanoma is safe and shows very promising activity. Propranolol 30 mg twice a day was selected as RP2D in addition to pembrolizumab based on safety, tolerability, and preliminary antitumor activity.

Translational Relevance

Nerves of the sympathetic nervous system, and β-adrenergic receptor signaling, are now known to play a role in cancer promotion and suppression of the immune system. The β2-receptor signaling pathway has been shown to create an immunosuppressive tumor microenvironment by decreasing CD8+ T cells and increasing the number and proliferation of myeloid-derived suppressor cells. Retrospective observational and prospective trials suggest a therapeutic role for β-blockers in patients with cancer. This is the first prospective phase I study combining pembrolizumab with a nonselective β-blocker, propranolol, to inhibit the stress pathway in patients with metastatic melanoma to determine safety and tolerability along with biological and correlative endpoints. The combination was tested in nine patients and found to be safe with objective response in seven of nine patients. These results strongly support our expanded phase II study investigating propranolol and pembrolizumab in patients with metastatic melanoma and additional studies of β2-adrenergic blockade in other advanced malignancies.

Introduction

Use of immune checkpoint inhibitors has revolutionized the treatment of melanoma. A recent 5-year follow-up of patients with treatment-naïve metastatic melanoma treated with pembrolizumab has shown an overall survival (OS) rate of greater than 40% (1). Similarly, the CheckMate 067 trial showed a 44% 5-year survival rate and 45% objective response rate with nivolumab monotherapy in untreated unresectable stage III or IV melanoma (2). Although response rates and OS are numerically higher with combination of anti–PD-1 and anti-CTLA4 checkpoint inhibitors, 59% of patients developed grade 3 or 4 treatment-related adverse events (TRAEs) with combination of nivolumab and ipilimumab, as opposed to 23% with nivolumab monotherapy (2). On the other hand, with pembrolizumab monotherapy, grade ≥3 TRAEs were seen in only 17% of patients (1). These data highlight an unmet need for better combination therapies using anti–PD-1 checkpoint inhibitors that result in improved outcome and manageable toxicity profiles.

Increased β-adrenergic receptor (β-AR) signaling, driven by sympathetic nerves of the autonomic nervous system, has been shown to increase tumor growth in mouse melanoma models. Several studies have shown that β-adrenergic blockade with nonselective β-blocker, propranolol, in mouse melanoma models decreases tumor growth and metastasis (3, 4). Propranolol exerts an antitumor effect by favorably modulating the tumor microenvironment (TME) by decreasing myeloid-derived suppressor cells (MDSCs) and increasing CD8+ T cells and natural killer cells in the TME (5). In addition, Bucsek and colleagues have shown a synergistic action of anti–PD-1 antibody and propranolol using a B16-OVA melanoma model in mice experiencing chronic stress. Stress conditions were created by housing mice under the standard housing temperature at 22°C required for experimental mouse colonies (4). In addition, prior research in murine models and healthy volunteers has shown that stress-induced sympathetic nervous system leads to increase in levels of proinflammatory cytokines inflammation (6–8), which may be a key biological mechanism of how stress affects health and promotes tumor progression. Pharmacologically blocking this sympathetic activation attenuates the inflammatory response to stress (6). These preclinical observations form the foundation of our clinical investigation of this combination therapy in patients with metastatic melanoma.

Multiple retrospective studies have shown an improvement in OS in patients with cancer treated with β-blockers (9–11). A prospective study in patients with early-stage melanoma treated with adjuvant propranolol reported an improvement in disease-free survival (12). In a retrospective study, Kokolus and colleagues have shown a statistically significant improvement in OS in patients with metastatic melanoma treated concurrently with nonselective β-blocker and immunotherapy compared with patients treated with immunotherapy alone. Interestingly, no benefit was seen in patients who were treated with β1-selective blockers (13). In our retrospective analysis, we did not see an improvement in OS in patients concurrently treated with β-blocker and checkpoint inhibitor, which could be due to a mix of different tumor histologies analyzed in the study, in addition to a small number of patients on nonselective β-blockers. No increase in grade ≥3 adverse events (AEs) was observed in this cohort (14).

On the basis of preclinical data showing synergistic activity of anti–PD-1 antibody and propranolol, and retrospective data showing efficacy and safety of this combination, we decided to prospectively evaluate the combination of propranolol and pembrolizumab in patients with metastatic melanoma. We hypothesized that the addition of the nonselective β-blocker, propranolol, to pembrolizumab may improve its antitumor efficacy by decreasing adrenergic stress, which is likely to be increased in patients with a new diagnosis of cancer.

Patients and Methods

Study population

Eligible patients were recruited from the melanoma clinic at Roswell Park Comprehensive Cancer Center (Roswell Park; Buffalo, NY) between January 2018 and August 2019. Eligibility criteria included adult patients (aged ≥ 18 years), Eastern Cooperative Oncology Group (ECOG) performance status of 0 (indicating no symptoms) or 1 (indicating mild symptoms) with treatment-naïve, histologically confirmed unresectable stage III or IV melanoma (any histologic subtype), with good organ function, and measurable disease on CT (preferred) or MRI scans per immune-modified RECIST (imRECIST) guidelines, and absence of symptomatic brain metastases. Key exclusion criteria were prior therapy with PD-1/PD-L1 inhibitors, chronic autoimmune disease or other immunodeficiency syndromes, contraindication to use of β-blockers (uncontrolled depression, grade III or IV heart failure, severe asthma or chronic obstructive pulmonary disease, uncontrolled type 1 or type 2 diabetes mellitus with HbA1C > 8.5 or fasting plasma glucose > 160 mg/dl, symptomatic peripheral arterial disease, or Raynaud syndrome), and current or past use of β-blockers or calcium channel blockers in the last 2 years.

The study was approved by the Institutional Review Board at Roswell Park (Buffalo, NY) and registered on the clinicaltrials.gov website (NCT03384836). Informed consent was obtained from all participants prior to enrollment. The study was performed in accordance with the Declaration of Helsinki and the International Council for Harmonisation/Good Clinical Practice guidelines.

Study design

This trial was an open-label, single-arm, nonrandomized, single-center, dose-finding phase Ib study. Dose escalation followed a “3 + 3” design, and no intrapatient dose escalation was allowed. Eligible patients were treated with standard-of-care pembrolizumab 200 mg i.v. every 3 weeks and progressively increasing propranolol doses from 10 mg (dose level 1), 20 mg (dose level 2) to 30 mg (dose level 3) twice a day, until 2 years on study or disease progression or dose-limiting toxicities (DLTs). A total of nine patients were accrued between January 2018 and August 2019. At the cut-off date of February 3, 2020, four patients continue to be on the study treatment per protocol.

Study objectives and endpoints

The primary objective of the study was to assess the safety and efficacy (overall response rate within 6 months of starting therapy) of combination of pembrolizumab with increasing doses of propranolol for unresectable stage III and metastatic melanoma, and to select the recommended phase II dose (RP2D) on the basis of safety, efficacy, and biomarker analyses.

Safety outcomes were assessed by physical examination, laboratory findings, vital signs, and electrocardiogram. AEs were graded by Common Terminology Criteria for Adverse Events (CTCAE) v4.03. No AEs due to propranolol doses of 10–30 mg twice a day were anticipated. Nevertheless, a serious AE/DLT due to propranolol was defined as any life-threatening AE (e.g., symptomatic bradycardia or symptomatic hypotension) that would mandate recruitment per the 3 + 3 design. Otherwise, a DLT was defined as grade 3 and higher immune-related AE (irAE), pneumonitis, colitis, hepatitis, nephritis, anemia, myositis, and cardiomyositis, as defined by CTCAE v4.03, new onset diabetic ketoacidosis, Guillain–Barre syndrome, or any other condition, which the investigator believed to be an immune-mediated AE and necessitated stopping therapy. Endocrinopathies were not included as DLTs, as the hormones will be replaced.

Objective response was defined as confirmed complete response or confirmed partial response (PR) among all treated patients with measurable disease at baseline.

The secondary objectives were to analyze efficacy as progression-free survival (PFS) and OS. PFS was measured from treatment initiation to time of disease progression or death, while OS was measured from the date of starting the treatment until date of death or censoring. Exploratory objectives included analysis of biomarkers over time on study.

Assessments

Patients were assessed for tumor response according to imRECIST every 12 weeks (±14 days) for the first 6 months, and then per physician discretion until confirmed disease progression or toxicities. Safety assessments occurred at each clinic visit.

Exploratory analyses

Baseline tumor tissues, archival or fresh biopsy, were analyzed. Participants underwent serial blood collection into heparin and EDTA tubes for analysis of several biomarkers in peripheral blood. Patients completed the validated perceived stress scale (PSS) questionnaire (15) at baseline and at additional timepoints to measure and quantify patient-reported stress-level perception. The results are reported as low stress (scores, 0–13), moderate stress (scores, 14–26), and high stress (scores, 27–40; ref. 15).

Tissue collection and analyses

Participants underwent tumor tissue collection at baseline for diagnosis, prior to study enrollment. Two of nine patients had a fresh biopsy and archival tissue was used for seven of nine patients. Formalin-fixed, paraffin-embedded tissues were sectioned at 4 μm for multispectral immunofluorescence staining with antibodies against the following markers: CD8 (Dako, clone CD8/144B, dilution 1:250), CD4 (Dako, clone 4B12, ready to use), Foxp3 (Abcam, clone 236A/E7, dilution 1:125), CD14 (Cell Marque, clone EPR3563, dilution 1:100), CD15 (Dako, clone Carb-3, dilution 1:50), PD-L1 (Abcam, clone SP142, dilution 1:100), TIM3 (Cell Signaling Technology, clone D5D5R, dilution 1:100), OX40 (Cell Signaling Technology, clone E9U7O, dilution 1:50), CTLA4 (BioCare, clone UMAB249, dilution 1:50), PD-1 (Cell Signaling Technology, clone EH33, dilution 1:100), LAG3 (Cell Signaling Technology, clone D2G4O, dilution 1:250), and DAPI. Multispectral staining was performed after antibodies were optimized for standard IHC and uniplex immunofluorescence staining. Slides were imaged using the Vectra Polaris Spectral Imaging System (PerkinElmer). Slides were initially scanned at 4 ×, visualized using Phenochart Viewer (PerkinElmer), and five tumor areas per case were selected for scanning at high resolution (20 ×). Each fluorophore from PerkinElmer Opal Kit was measured using a separate filter cube corresponding to its emission wavelength. The images were unmixed using a spectral library and individual fluorophores were separated with InForm software. The immune cell populations were quantified using cell segmentation and phenotype cell tool from InForm 1.1 (PerkinElmer). Threshold for positive staining and accuracy of phenotypes were confirmed by pathologist supervision (A.K. Witkiewicz). The individual markers from the panel were quantified and plotted as the average of the positive staining cells across the regions of interest.

Flow cytometry of peripheral blood

Flow cytometry was used to quantify MDSC and regulatory T cell (Treg) populations in freshly isolated peripheral blood samples from heparinized tubes. An eight-color panel comprising CD11b FITC, CD16 PE, CD45 PerCP, CD33 PECy7, HLADR APC, CD14 APCH7, and CD15 V450; a lineage dump consisting of CD3, CD19, and CD56 (all conjugated to BV510) was used to measure early precursors MDSC (eMDSC), monocytic MDSC (mMDSC), and polymorphonuclear MDSC (PMN-MDSC) subsets (panel 1). Separately, a six-color panel comprising of CD8 FITC, CD25 PE, CD4 PerCP, CD3 PECy7, CD45 APCH7, and CD127 BV421 was employed to measure T-cell subsets (panel 2). WinList software (version 8.0; Verity Software House) was employed for the analysis of flow cytometric data. Analyzed data were reported as absolute cell count (cells/μL), or separately as the percentage of CD45+ events. MDSC populations were quantified according to the phenotypic definitions established by Bronte and colleagues (16). In brief, to quantify eMDSCs, mononuclear cells (defined on the basis of their CD45 expression profile and light scatter characteristics) were sequentially gated to bivariate plots of HLADR versus DUMP, CD14 versus CD15, and CD33 versus CD11b; where eMDSCs were further defined as HLADRlow/−, DUMP−, CD14−, CD15−, CD33+, and CD11b+. To quantify mMDSCs, mononuclear cells were sequentially gated to bivariate plots of CD11b versus CD15 and HLADR versus CD14; where mMDSC were further defined as CD11b+, CD15−, HLADRlow/−, and CD14+. To quantify PMN-MDSCs, mononuclear cells were sequentially gated to bivariate plots of CD14 versus CD15 and CD11b versus SSC-A; where PMN-MDSCs were further defined as CD14−, CD15+, and CD11b+. Separately, T-cell subsets were quantified using panel 2. Th cells were defined as CD3+, CD4+, and CD8− and cytotoxic T cells were defined as CD3+, CD4−, and CD8+. To quantify Tregs, CD3+, CD4+, and CD8− T cells were gated to a bivariate plot of CD25 versus CD127; where Tregs were further defined as CD25+ and CD127(dim).

Chemokines/cytokines in peripheral blood

Plasma was collected from EDTA blood and stored as aliquots at −80°C. 29-plex MILLIPLEX MAP human cytokine/chemokine magnetic bead panel 96-well plate assay was used to examine blood plasma levels of cytokines and chemokines. Wash buffer, sheath fluid, serum matrix, samples, and standards were prepared in accordance with the manufacturer's protocol. The resultant data were analyzed using Upstate BeadView software for median fluorescence intensity using a five-parameter logistic curve-fitting method to calculate analyte concentrations in both samples and control wells.

Statistical analysis

One of the primary objectives was to identify an RP2D based on safety, efficacy, and the biomarker profile. As such, a standard 3 + 3 design was considered, with three dose levels, and requiring up to 18 subjects. Patients receiving at least one dose of pembrolizumab were evaluable for response and safety.

In the primary analysis, AEs and objective response were summarized by dose level using frequencies and relative frequencies.

For intradose analysis, peripheral blood biomarkers were summarized by dose level and timepoint using mean plots (± SE). For intradose-level comparisons, the markers were modeled as a function of timepoint and a random subject effect using a linear mixed model. An F test about the main effect of time was used to evaluate whether marker expression changes over time. In addition, the mean level at each timepoint was compared with baseline using Dunnett adjusted tests. For interdose-level comparisons, percent change was calculated from baseline for each biomarker. The mean percent change was compared between dose levels using an ANOVA model, with pairwise comparisons made using a Tukey adjustment. All model assumptions were verified graphically, and transformations were applied as appropriate. All analyses were completed in SAS v9.4 at a significance level of 0.05.

Results

Patient characteristics

As of the data cut-off date of February 3, 2020, nine patients with metastatic cutaneous melanoma that were treatment-naïve to PD-1/PD-L1 and CTLA-4 inhibitors completed enrollment for the phase I safety study. The median age of patients on the study was 65 years (35–76). Six patients were females (67%) and all patients were white. At baseline, six patients had an ECOG performance score of 0 (67%), five patients had M1c disease (56%), and three patients had elevated lactate dehydrogenase levels (LDH, 33%). The baseline PSS score ranged from 6 to 30, with a median score of 13. Four of nine patients (44%) remain on study treatment. Primary reasons for study discontinuation were AEs in two patients (22%) and disease progression in three patients (33%). Baseline patient and disease characteristics are summarized in Table 1.

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Table 1.

Baseline characteristics of the patient population.

Safety and tolerability

TRAEs occurred in all nine patients. Al but one patient had TRAEs that were grade 2 or lower. The most commonly reported TRAEs were fatigue, rash, and vitiligo, which occurred in four of nine (44%) patients.

Serious TRAEs leading to discontinuation of therapy occurred in two patients, both in the 20-mg twice-a-day cohort: hemophagocytic lymphohistiocytosis and labyrinthitis. TRAEs are shown in Table 2. There were no deaths on study treatment. Two grade ≥3 AEs were reported in one patient. That patient developed a grade 3 increase in alanine aminotransferases (ALTs), which was treated with oral prednisone. Subsequently, he was found to have hepatitis C for which he was treated successfully. Later, he had a hospital admission complicated by necrotizing fasciitis, deep vein thrombosis, and hemophagocytic lymphohistiocytosis. Details of all AEs are shown Supplementary Table S1.

View this table:
Table 2.

TRAEs while on the study.

No DLTs were observed at any of the three dose levels. Hence, propranolol at all three dose levels was considered for RP2D.

Preliminary antitumor activity

By the cut-off date of February 3, 2020, the median follow-up was 15.6 (range, 5.4–24.2) months. The median number of pembrolizumab cycles received were 7.5 (2–32). Objective responses were noted at all three dose levels (Table 3). The objective response rate was seven of nine (78%) on the study.

View this table:
Table 3.

Best responses per imRECIST and current status of patients on study as of cut-off date.

Figure 1 shows the spider plot for tumor response for all the nine patients on the study (P1–P9). In dose level 1, two patients experienced tumor response, and one patient had stable disease (SD) as best response. The patient with SD (P1) had a mixed response (increase in size of axillary lymph node and decrease in size of subcutaneous lesions), ultimately coming off study to receive radiation to the growing axillary lymph node and progressing per imRECIST. The patient continues to remain on combination of pembrolizumab and propranolol off study (total follow-up of 24.2 months).

Figure 1.
Figure 1.

Spider plot of the change in the sum of tumor diameters over time for the nine evaluable patients on study. Zero on the x-axis corresponds to the time of the baseline scan and percentage change in tumor size from baseline is shown on the y-axis.

In dose level 2, two patients had a PR, and one patient came off study due to the development of rapid disease progression. Both patients with PR discontinued combination therapy because of toxicity. One patient has maintained PR (total follow-up of 18.9 months), whereas the other patient had progressive disease (PD) and underwent metastasectomy for the residual metastatic lesion.

In dose level 3, all three patients experienced PR and continue on study treatment. As of the cut-off date, four patients continue on treatment per protocol. One patient died because of disease progression.

PSS

We hypothesized that patients reporting increased stress would be more likely to respond due to blockade of the elevated β2-AR signaling. At baseline, five of nine patients had low PSS scores, three of nine had moderate scores, and one of nine had a high score. Responses were seen at all PSS score levels (Supplementary Fig. S1A). We observed a trend toward decrease in PSS scores over time in dose levels 1 and 3. In dose level 2, although subsequent PSS scores were lower than baseline, a similar trend could not be conclusively established because of lack of sufficient data points in that cohort (Supplementary Fig. S1B).

Chemokine and cytokine analysis

Intradose comparison

In dose level 1, 3- (P = 0.002), 6- (P = 0.008), 9- (P = 0.023), and 12- (P = 0.018) levels of IP-10 were significantly higher than at baseline. Immunosuppressive chemokine, eotaxin, showed significant time effect in dose level 2 (P = 0.04) with a large increase in expression around 12 weeks compared with baseline (2/3 patients). In addition, there was a significant decrease of immunostimulatory cytokine, TNFβ, at 12 weeks compared with baseline in dose level 2 (P = 0.003; 1/3 patients). Supplementary Fig. S2A shows variation in levels of IP-10, eotaxin, and TNFβ in the three dose levels.

Interdose comparison

There was a decrease in expression of IL12p70 in dose level 3 compared with dose levels 1 (P = 0.007) and 2 (P = 0.012) at week 6 (Supplementary Fig. S2C)

Responder versus nonresponder

At week 3, compared with baseline, IFNγ was increased in dose level 1 group and in two of three patients in dose level 3 (all these were responders) and decreased in both nonresponders. Interestingly, IL6 decreased in five of six responders (value was not available for one responder) and increased in one nonresponder (although decreased in nonresponder with mixed response; P1). An increase in IP-10 was observed among all patients. Figure 2A shows the heatmap of chemokine changes in responders and nonresponders. Supplementary Table S2A shows the absolute quantitative change in chemokines from baseline to week 3 on study.

Figure 2.
Figure 2.

Heatmap showing changes (increase, decrease, or no change) in chemokines and cytokines (A) and cellular fractions (B) at 3 weeks compared with baseline while on study treatment at different dose levels of propranolol separated for responders (P2, 3, 4, 5, 7, 8, and 9) and nonresponders (P1 and 6). Percentage of cellular fractions refers to the absolute number expressed as a percentage of total CD45+ cells.

Flow cytometry

Intradose comparison

Supplementary Fig. S2B shows variation in CD8+ T-cell %, mMDSC %, Treg %, PMN-MDSC % (expressed as a percentage of total CD45+ cells), and ratio of CD8+T cell/PMN-MDSC in the three dose levels. At all dose levels, there was a trend toward an initial increase in CD8+ T cells/total CD45+ cells (CD8+T-cell %) until week 3. There was also a trend of early increase in the mMDSC % and Treg % at all dose levels. Interestingly, in dose level 1, 30-week mMDSC % and PMN-MDSC % were significantly higher (P < 0.01 and P < 0.01, respectively), whereas 30-week ratio of CD8+/PMN-MDSC was lower (P = 0.01) when compared with baseline (Supplementary Fig. S2B). A trend in early decrease in PMN-MDSC % was seen in the initial 3 weeks in dose levels 2 and 3.

Interdose comparison

Relative to baseline, significant decrease in PMN-MDSC % at 3 weeks was observed for dose level 2 compared with dose levels 1 (P = 0.004) and 3 (P = 0.007). Relative to baseline, highest increase in CD8+ T-cell % and ratio of CD8+T cell/Treg was seen in dose level 3, although not statistically significant (Supplementary Fig. S2C).

Responder versus nonresponder

There was significant heterogeneity among responders at different dose levels. Interestingly, at week 3, the ratio of CD8+T cell/mMDSC increased compared with baseline in three of three responders in dose level 3 and decreased in nonresponders. An increase in CD8+ T-cell % compared with baseline was seen in three of three responders in dose level 3 versus decrease in nonresponder (although increased in a nonresponder with mixed response; P1). An increase in Treg % was seen in all patients. Figure 2B shows the heatmap of cellular changes in responders and nonresponders. Supplementary Table S2B shows the quantitative change in peripheral blood cellular fractions from baseline to week 3 on study.

Tissue biomarkers

As shown in Supplementary Fig. S3, multispectral staining revealed marked variability in the composition and the distribution of immune infiltrate at the baseline. To further characterize the CD8+T cells and analyze impact of exhaustion markers on response to treatment (17), multispectral analysis of T-cell activation and exhaustion markers was performed using antibodies for CD8, OX40, CTLA4, PD1, LAG3, and TIM3. Representative multiplex and monoplex images are shown (Supplementary Fig. S4A). Phenotyping was performed using InForm software to delineate activated T cells, cytotoxic T cells, partially exhausted T cells, and exhausted T cells, with the underlying marker analysis illustrated for a representative tumor (Supplementary Fig. 4B) at baseline. Supplementary Table S3 shows the percentage of different cellular fractions in the study patients in the regions of interest selected for the analysis. Nonresponders (P1 and P6) had a lower number of CD8+ cytotoxic T cells, however, three of the patients who responded to the therapy had comparable levels of CD8+ cytotoxic T cells, indicating that in a subset of patients, antitumor immune response can be activated independently of the baseline level. The number of mMDSC was highly variable among responders and nonresponders. The patient with rapid disease progression had the highest percentage of PMN-MDSCs (P6). Nonresponders exhibited high expression of PD-L1 on mMDSCs or PMN-MDSCs relative to patients who benefited from the therapy. PD-L1+ melanoma cells varied from 0.34% to 29.34% and did correlate with the response. Other cellular fractions or ratios, as shown in Supplementary Table S3, were not predictive of clinical response; however, our analyses is limited by the small size of a phase I study.

Notably, one of the nonresponders (P6), had a clear-cell morphology with minimal immune infiltration (Supplementary Fig. S5). Interestingly, this patient also had <1% PD-L1+ melanoma cells, a higher infiltration of PMN-MDSCs, and a lower CD8+ T-cell infiltration compared with other patients.

RP2D

Propranolol 30 mg twice a day was chosen as the RP2D in this study in combination with pembrolizumab as there were no DLTs at this level, preliminary antitumor efficacy was observed in all three patients at this dose level, and these patients demonstrated a trend toward higher CD8+ T-cell %, and ratios of CD8+ T cell/Treg and CD8+ T cell/PMN-MDSC. These data are intriguing, however, due to the small size and significant heterogeneity in biomarkers, any statistically sound and meaningful interpretation of biomarkers to decide a RP2D is difficult.

Discussion

Our phase I study shows that the combination of propranolol and pembrolizumab is safe in treatment-naïve metastatic melanoma. As the dose–effect relationship of propranolol on immune regulation is not well defined, another objective of the study was to select the RP2D of propranolol in combination with pembrolizumab based on toxicity, response, and biomarker analyses. No DLTs were observed on the study. Observed frequency of AEs was not higher than expected with pembrolizumab monotherapy alone. However, two patients developed rare toxicities: hemophagocytic lymphohistiocytosis and ototoxicity. One patient developed hemophagocytic lymphohistiocytosis after three cycles of pembrolizumab, during admission for necrotizing fasciitis from methicillin-resistant Staphylococcus aureus. Although rare, cases of hemophagocytic lymphohistiocytosis from pembrolizumab monotherapy have been reported in literature (18, 19). Staphylococcus aureus is a known risk factor for secondary hemophagocytic lymphohistiocytosis and could have been a contributing cause (20). Hemophagocytic lymphohistiocytosis was resolved with steroids, without requirement of additional immunosuppression. No further episodes of hemophagocytic lymphohistiocytosis relapse were observed until last follow-up. Another patient developed grade 2 ototoxicity (hearing loss/labyrinthitis) after two cycles of pembrolizumab, which was treated with intratympanic and oral steroids. This patient had no evidence of leptomeningeal disease, but still has residual hearing loss. The mechanism(s) underlying these rare irAE remains unclear. β-ARs present in the inner ear epithelium play an important role in ion transportation (21). Some β-blockers have been implicated in hearing loss (22). In addition, in rats, anti–PD-1 has been shown to be directly toxic to hair cell and organ of Corti (23). It is also interesting to note that the inner ear is rich in melanocytes. Vogt-Koyanagi-Harada disease, an autoimmune disease targeting melanocytes frequently involves the inner ear (24). Ototoxicity in our patient could also be due to a cross-reactive autoimmune response of the patient's T cells to melanocytes in the inner ear. Autoimmune hearing loss, although rare, has been reported as a toxicity of pembrolizumab monotherapy (25). With regard to hemophagocytic lymphohistiocytosis observed in 1 patient, we note that hemophagocytic lymphohistiocytosis was observed in the context of a severe Staphylococcus aureus wound infection. Staphylococcus aureus has also been associated with secondary hemophagocytic lymphohistiocytosis (26).

In treatment-naïve patients, the objective response rate has been reported as 46% with pembrolizumab monotherapy using immune-related response criteria, and median PFS as 11.6 months (1). In our study, responses were observed at all three dose levels of propranolol with an objective response in seven of nine patients (78%). Five of seven patients continue to demonstrate maintenance of tumor response. After a median follow-up of 15.6 months, two of nine (22%) patients have gone on to receive second-line systemic therapy. Median PFS and OS have not been reached. Four patients continue on study treatment per protocol, and another patient continues on combination treatment off study.

Interestingly, compared with baseline, we saw an early increase in the levels of mMDSCs in peripheral blood of patients at several timepoints and at all doses. Low baseline blood mMDSC levels have been shown to be predictive of response to anti–CTLA-4 therapy (27). However, the prognostic value and kinetics of mMDSCs during anti–PD-1 therapy remain unclear. Similar to our findings, another study of patients with metastatic urothelial cancer treated with pembrolizumab reported an increase in mMDSCs during therapy (28). At week 3, we noted an increase in IFNγ (4/7 patients) and a decrease in IL6 (5/6 patients) in responders. IL6 also decreased in the patient with mixed response. These findings are consistent with our findings in 4T1 and AT3 mammary mouse models, where reducing adrenergic stress or use of β2-AR−/− mice was associated with decreased plasma levels of protumor cytokines and increased antitumor cytokines, decrease in IL6, and increase in IFNγ, yet no significant changes in IP-10 were observed (5). The increase in IP-10 as seen in this phase I trial cohort could be an effect of pembrolizumab. This raises an interesting question whether treatment-induced biomarkers could predict outcomes and help select a bioequivalent propranolol dose in humans; however, sample size in each cohort was too small for any meaningful biomarker interpretation. Although these peripheral blood findings are interesting, differences induced in peripheral blood do not always display parallel changes intratumorally (29). The observation that the 1 patient with rapid disease progression had little T-cell infiltrate also suggests that serial tumor biopsies on-treatment could provide important information relative to disease response and mechanisms of propranolol action in the TME.

Responses were observed irrespective of baseline PSS score levels. Interestingly, although a new cancer diagnosis is considered a stressful event (30–33), a majority of the patients had a low PSS score. Over a period of time, we observed a decrease in median PSS scores may have been associated with development of coping skills while undergoing treatment, or a positive experience of disease response, which occurred in most patients. Ultimately, an assessment of whether β2-AR blockade could influence outcomes in patient reporting moderate or high stress scores is limited by the small number of patients and a lack of control group without propranolol intervention.

The primary intent of this phase I dose-escalation study was to investigate the safety of combining pembrolizumab with propranolol, that it did not increase the expected toxicities compared with pembrolizumab monotherapy alone, and to ensure that the combination does not compromise the historic efficacy of pembrolizumab monotherapy. Lower doses of propranolol were used because of lack of prospective evidence that the cardiovascular effects of pan-β blockade correlate with pharmacodynamics impact of propranolol on immune pathways. We note the provocative findings from Hiller and colleagues, where patients with breast cancer received intrapatient dose escalation of propranolol from 80 to 160 mg daily that, patients with evidence of clinical β-blockade had higher tumor immune infiltration with CD68+ macrophages and CD8+ T cells (34). However, this was a post hoc analysis and the clinical trial was not designed to answer the question about the appropriate dose of β-blocker to achieve this effect on immune cells. Because our clinical trial focused on studying the safety of this combination, we used lower doses of propranolol to ensure that there are no increased toxicities or compromise of efficacy compared with pembrolizumab alone. Imbedded in our ongoing phase II study is an evaluation of heart rate–induced exercise as a biomarker of outcome.

Our study has several caveats that must be addressed in future, larger studies. Because this was a phase I study with the primary endpoint to determine safety of the combination, the biomarker analyses could only be exploratory in nature, and the study was not designed to evaluate the impact of treatment on TME. Because of lack of a control group for comparison, we could not attribute any immunologic effects specifically to propranolol alone in the combination. Interpretation of some immunologic biomarkers was limited because of lack of longitudinal samples for some patients as a result of disease progression or toxicities. The study design, sample size, and objectives are consistent with phase I oncology trials and was not designed as a formal assessment of efficacy. The response rate noted met our predetermined goal of not negatively impacting the single-agent activity of pembrolizumab. We recognize that the remarkable response rate in this study is provocative and should be interpreted with caution until the larger multicenter phase II study, currently ongoing, is completed.

To our knowledge, this effort is the first prospective clinical trial to show that the combination of propranolol with pembrolizumab is safe, and additionally suggests preliminary synergistic antitumor activity in treatment-naïve metastatic melanoma. On the basis of safety, response, and biomarkers, propranolol at a 30 mg twice a day dose was chosen as the RP2D dose. A larger phase II, multicenter, single-arm study is ongoing to confirm and validate the findings from this study.

Authors' Disclosures

S. Gandhi reports grants and other from NCI during the conduct of the study. P. Funchain reports grants from Pfizer and personal fees from Eisai outside the submitted work. I. Puzanov reports personal fees from Merck, Amgen, and BMS outside the submitted work. M.S. Ernstoff reports grants and other from NCI during the conduct of the study, personal fees and other from Alkermes, personal fees from BMS, ImmuNext, and EMD Serono, and other from Merck outside the submitted work. No disclosures were reported by the other authors.

Disclaimer

The authors take full responsibility for the design of the study, the collection of the data, the analysis and interpretation of the data, the decision to submit the article for publication, and the writing of the article. This article was prepared while M.S. Ernstoff was employed at Roswell Park Comprehensive Cancer Center. The opinions expressed in this article are the author's own and do not reflect the view of the NIH, the Department of Health and Human Services, or the U.S. Government. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Authors' Contributions

S. Gandhi: Conceptualization, data curation, formal analysis, funding acquisition, writing-original draft, project administration, writing-review and editing. M.R. Pandey: Conceptualization, data curation, formal analysis, funding acquisition, writing-original draft, writing-review and editing. K. Attwood: Formal analysis, writing-original draft, writing-review and editing. W. Ji: Formal analysis, writing-review and editing. A.K. Witkiewicz: Formal analysis, writing-original draft, writing-review and editing. E.S. Knudsen: Formal analysis, writing-review and editing. C. Allen: Investigation, writing-original draft, writing-review and editing. J.D. Tario: Investigation, writing-original draft, writing-review and editing. P. Wallace: Investigation, writing-review and editing. C.D. Cedeno: Investigation, writing-review and editing. M. Levis: Resources, writing-original draft, writing-review and editing. S. Stack: Resources, writing-original draft, writing-review and editing. P. Funchain: Writing-review and editing. J.J. Drabick: Writing-review and editing. M.J. Bucsek: Conceptualization, writing-review and editing. I. Puzanov: Writing-review and editing. H. Mohammadpour: Formal analysis, writing-review and editing. E.A. Repasky: Conceptualization, formal analysis, writing-original draft, writing-review and editing. M.S. Ernstoff: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, writing-original draft, project administration, writing-review and editing.

Acknowledgments

We thank the patients, their families, and the study personnel for participating in this study. The research reported in this article was supported by the Roswell Park Alliance Foundation grant (to S. Gandhi), Melanoma Fund (generous gifts from patients, to M.S. Ernstoff), Roswell Park Comprehensive Cancer Center, NCI grant P30CA016056, NCI grant R01CA205246 (to E.A. Repasky), Harry J Lloyd Charitable Trust (to E.A. Repasky), and National Center for Advancing Translational Sciences of the NIH under award Nos. 5KL2TR001413-05 and UL1TR001412-05 (to S. Gandhi).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

  • Clin Cancer Res 2021;27:87–95

  • Received June 18, 2020.
  • Revision received September 9, 2020.
  • Accepted October 21, 2020.
  • Published first October 30, 2020.

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Phase I Clinical Trial of Combination Propranolol and Pembrolizumab in Locally Advanced and Metastatic Melanoma: Safety, Tolerability, and Preliminary Evidence of Antitumor Activity
Shipra Gandhi, Manu R. Pandey, Kristopher Attwood, Wenyan Ji, Agnieszka K. Witkiewicz, Erik S. Knudsen, Cheryl Allen, Joseph D. Tario, Paul K. Wallace, Carlos D. Cedeno, Maria Levis, Suzanne Stack, Pauline Funchain, Joseph J. Drabick, Mark J. Bucsek, Igor Puzanov, Hemn Mohammadpour, Elizabeth A. Repasky and Marc S. Ernstoff
Clin Cancer Res January 1 2021 (27) (1) 87-95; DOI: 10.1158/1078-0432.CCR-20-2381
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